Tautomer-Selective Fluorescence Spectroscopy of Oxyluciferin Anions

Bioluminescence in fireflies and related insects arises
as emission
from the fluorophore oxyluciferin, yet the color of the emission in
these insects can range from red to green. The chromophore’s
microenvironment or multiple tautomeric forms may be responsible for
the color tuning; however, these effects are difficult to separate
in condensed phases. To investigate the role of oxyluciferin tautomerization
in the color tuning mechanism, gas-phase spectroscopy eliminates solvent
effects and allows us to study the fluorescence from individual tautomers.
Using a home-built mass-spectrometry setup with a cylindrical ion
trap cooled with liquid nitrogen, we measure fluorescence from the
enol-locked form of oxyluciferin in the gas phase and characterize
the photophysics of both keto and enol forms. At 100 K, the enol-locked
form has an emission maximum of 564 ± 1 nm, coinciding with a
previously reported assignment in oxyluciferin. We measure the absorption
spectrum and find a maximum at 560.5 ± 0.5 nm, which implies
a Stokes shift of 110 cm–1. The absorption spectrum
is compared to Franck–Condon simulated spectra that identify
one dominant vibrational mode in the transition. Additionally, we
ultimately separated the emission by the enol and keto forms present
in the trap by selectively exciting each form. We demonstrate that
fluorescence measured close to the 0–0 transition limits the
reheating of the ions, thereby providing the coldest ions and therefore
the narrowest emission spectra. These experimental data are also crucial
benchmarks for computational studies, offering actual emission spectra
in the gas phase for both tautomeric forms. Thus, our findings serve
as essential reference points for excited-state calculations aimed
at understanding the color tuning mechanism of bioluminescence computationally.